Interleukin-21 (IL-21) is a recently identified type 1 cytokine, which is secreted as a 133-amino acid protein by activated CD4+ T cells (Parrish-Novak, J. et al., Nature 408, 57-63 (2000)). The IL-21 cytokine has been demonstrated to possess potent stimulatory effects on the proliferation, differentiation and activation of several classes of haematopoietic cells including B-cells, T-cells and NK-cells. The biological effects of IL-21 are mediated via activation of the IL-21 receptor complex, which is composed of an IL-21 private receptor chain (IL-21Rα) in complex with the common gamma chain (γc), which similarly constitutes an essential component of the signalling receptor complex of the cytokines IL-2, IL-4, IL-7, IL-9, and IL-15. These cytokines thus constitute a subfamily referred to as common gamma chain cytokines, with IL-21 being the most recently added member.
Within the common gamma chain family of cytokines, high resolution structural information has been obtained through X-ray crystallography and NMR spectroscopy for IL-2 and IL-4 (Brandhuber, B. J. et al., Science 238, 1707-1709 (1987), Mott, H. R. et al., Journal of Molecular Biology 247, 979-994 (1995), Powers, R. et al., Science 256, 1673-1677 (1992), Wlodaver, A. et al., Febs Letters 309, 59-64 (1992). It is apparent from these studies that IL-2 and IL-4 along with other type 1 cytokines, including IL-1β, IL-2, IL-4, and GM-CSF, share a common overall topology in their structures in spite of a distant homology in primary sequence. The common structural motif of these proteins consists of a central four-helical bundle, arranged in an up-up-down-down topology, connected by loops which are characterized by a high degree of structural freedom, a considerable difference in loop length, and variation in the number, and positioning, of stabilizing disulfide bridges. In the IL-21 amino acid sequence as shown in SEQ ID No. 1 (a 162 aa long polypeptide), helix A is defined by amino acid residues 41-56; helix B by amino acid residues 69-84; helix C by amino acid residues 92-105; and helix D by amino acid residues 135-148.
Crystal structures have also been reported for IL-2 and IL-4 in complex with the corresponding private chains and, in the case of IL-2, the common gamma chain (Wang, X. Q. et al., Science 310, 1159-1163 (2005), Hage, T. et al., Cell 97, 271-281 (1999)). IL-2 is distinct from both IL-4 and IL-21 by having two private receptor chains, IL-2Rα and IL-2Rβ, where IL-2Rβ is homologous to IL-4Rα and IL-21Rα. Only minor structural differences are observed between the free and receptor bound forms of IL-2 and IL-4 indicating that only slight structural changes occur for these cytokines upon complex formation. These studies accurately identify the residues of the cytokines involved in receptor binding, and closely mirror earlier results obtained from mutagenesis studies.
IL-4 antagonists have been designed by making variants for which binding to γc has been abolished while preserving binding to the private receptor chain. This was accomplished by a double mutation [R121D, Y124D] in helix D (Tony, H. P. et al., European Journal of Biochemistry 225, 659-665 (1994)). The IL-4 epitope for γc binding have been further explored by biacore analyses with IL-4 variants (Zhang, J. L. et al., European Journal of Biochemistry 269, 1490-1499 (2002). Recently, it has been shown that IL-4 and IL-21 bind to partially overlapping epitopes of γc (Zhang, J. L. et al., Biochemical and Biophysical Research Communications 300, 291-296 (2003)).
By analogy to the IL-4 antagonist ([R121D, Y124D]-IL-4), IL-21 variants with antagonistic properties have been generated by mutation of residues in helix D corresponding to R121 and Y124 in IL-4 (WO2003040313).
Both IL-21 agonism and antagonism have thus been implicated as a potentially useful mechanism for treating diseases and disorders. Generating IL-21 variants having modulated activity can be a useful tool in order to elucidate more about such diseases and disorders and may present potential targets for drug development. As such, there is a continuing need for IL-21-variants and a method for designing such.